Efficient cultivation conditions for human limbal epithelial cells.
To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.
Kim
Mee Kum
MK
Department of Ophthalmology, Seoul National University College of Medicine, and Seoul ARtificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea.
Lee
Jae Lim
JL
Oh
Joo Youn
JY
Shin
Mi Sun
MS
Shin
Kyeong Seon
KS
Wee
Won Ryang
WR
Lee
Jin Hak
JH
Park
Ki Sook
KS
Son
Young Sook
YS
D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D003584:Cytological Techniques; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D053551:Keratin-12 / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D015534:Trans-Activators / Q000378:metabolism
D017931:DNA Primers; D053551:Keratin-12; D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse